The following invention relates to a process for oxidising alkyl groups attached directly or via a linker, to a sulfonamide moiety by the use of a cytochrome P450 enzyme.
There are a large number of structurally complex molecules of interest to medicinal chemists, which possess a hydroxylated or carboxylated alkyl group, attached directly or via a linker, to a sulfonamide group. The synthesis of these molecules is complicated by the synthetic steps necessary to hydroxylate or carboxylate such alkyl groups whilst avoiding-undesirable side reactions.
This problem has been solved by the process of the present invention, which teaches the use of a cytochrome P450 enzyme to selectively oxidise an alkyl group attached directly or via a linker to a sulfonamide group. The process which is specific and tolerant of other functional groups in the molecule allows the synthetic chemist to introduce a hydroxy or carboxy group into the synthesis of a molecule at a late stage, in a one step process. This reduces the overall number of steps required to synthesise medicinally important molecules and so improves the efficiency of their preparation.
The term xe2x80x98Cytochrome P450xe2x80x99 is used to describe a superfamily of hemoprotein enzymes. They are characterised by the absorption band of their FeIIxe2x80x94CO complex form which has an absorption maxima at 447-452 nm, indicative of a thiolate ligated hemoprotein. They are also called heme-thiolate proteins, though this category is much wider than just cytochrome P450. Cytochrome P450 appear to have diversified from a common ancestor and they may be found in almost all forms of living organism, from animals and plants to fungi and bacteria, where they carry out the role of an oxygenase.
Cytochrome P450 catalysed reactions are known to produce a range of metabolites. The reactions may usually be classified as one of three types:
a) Hydroxylation, the insertion of an oxygen atom between the H atom and some other heavier atom such as carbon or nitrogen,
b) Epoxidation, the addition of an oxygen atom to a carbon-carbon double bond,
c) Heteroatom oxidation, the addition of an oxygen atom to the electron pair on a heteroatom.
They are also known to catalyse reduction reactions. Useful reviews of the cytochrome P450 enzyme are provided in Handbook of Drug metabolism, Ed. Thomas Woolf, Publisher Marcel Decker, ISBN 0824702298, March 1999, chap 4, p109 and by T. Omura, Biochemical and Biophysical Research Communications, 1999, 266, 690.
R. A. Johnson et al., Biooganic Chemistry, 1973, 2, 99, discloses the treatment of a number of dialkylbenzenes with the P450 enzyme containing microorganism Sporotrichum sulfurescens, to form an alcohol on the alkyl side chain.
The abstract of JP-60258173 discloses the synthesis of an xcex1-(3-t-butyl-5-hydroxy-t-butylbenzylidene)butyrolactone by microbial hydroxylation of the corresponding t-butyl precursor. Suitable microorganisms for the transformation are those from the Mucor and Aspergillus strains.
C. Cerniglia et al., Applied and Environmental Microbiology, 1984, 47, 111, discloses the transformation of 1- and 2-methylnaphthalene by Cunninghamella elegans into the corresponding hydroxymethyl derivative. A small amount of further metabolites were also isolated in which the product had been further oxidised to the carboxylic acid or where hydroxylation of the ring system had taken place.
H. L. Holland et al., Tetrahedron Asymm., 1994, 5, 1241, teaches the oxidation of chiral para substituted alkyl benzyl sulfides to the sulfoxide. In cases where the para substituent was i-propyl or t-butyl, hydroxylation at the terminal methyl of the alkyl group was seen, as well as oxidation of the sulfide. In the case of the i-propyl group, the corresponding sulfone was also seen. These transformations were conducted using the microorganism Helminthosporium NRRL-4671.
H. Schwartz et al., Appl. Microbiol. Biotechnol., 1994, 44, 731, investigates the microbial oxidation of ebastine to carebastine. Of 15 microorganisms examined, only the Cunninghamella strains provided the desired biotransformation.
WO-A-99/47693 discloses a method for making fexofenadine from terfenadine by a biotransformation performed using a microorganism culture of the genus Streptomyces at a pH ranging between 5 and 8.
The oxidation by a cytochrome P450 enzyme of alkyl groups attached directly, or via a linker, to a sulfonamide moiety, is new. Sulfonamides are known to exhibit antimicrobial activity. Accordingly, molecules containing a sulfonamide group have not been viewed as suitable candidates for use in a biotransformation process as it was thought they would kill any microorganism used. Surprisingly, it has now been found that molecules containing a sulfonamide group may be used in certain oxidative biotransformations.
According to a first aspect of the present invention there is provided a process for synthesising the compounds of formula (I)
Rxe2x80x94NHSO2xe2x80x94Xxe2x80x94Yxe2x80x94Zxe2x80x83xe2x80x83(I)
wherein
R is an organic radical;
X is selected from
a) a 5- or 6-membered monocyclic aromatic ring optionally containing one or two heteroatoms, each independently selected from O, N and S;
b) a C1-C6 alkylene group, straight chain or branched chain; and
c) a direct link;
Y is xe2x80x94C(CH3)2xe2x80x94 or xe2x80x94CH(CH3)xe2x80x94; and
Z is xe2x80x94CH2OH or xe2x80x94COOH;
which comprises oxidising a compound of formula (II)
xe2x80x83Rxe2x80x94NHSO2xe2x80x94Xxe2x80x94Yxe2x80x94CH3xe2x80x83xe2x80x83(II)
wherein
R, X and Y are as defined above, with a cytochrome P450 enzyme.
The process described may be used to oxidise a suitable alkyl group to the corresponding hydroxy or carboxy derivative. These oxidations proceed from the alkyl to the hydroxy and in the latter case, onwards from the hydroxy to the corresponding carboxylic acid. It will be appreciated that by careful manipulation of the reaction conditions, one may selectively isolate a product at the desired oxidation level and maximise the yield of the desired product. The oxidation may be stopped at the hydroxy derivative or allowed to continue onwards to the carboxylic acid. To produce the corresponding carboxylic acid no modification to the process is required other than that the process be given sufficient time to further oxidise the substrate. It should be noted that the nature of the R substituent may influence the speed of the reaction and hence the yields of the desired products. Whilst all P450 enzymes are considered suitable for conducting the disclosed invention, certain microorganisms will be particularly suitable for specific levels of oxidation.
This transformation is effected by a cytochrome P450 enzyme. Particularly favoured are microorganisms which contain the P450 enzyme. Preferred microorganisms are unicellular bacteria, exemplified by species such as Escherichia coli, filamentous bacteria exemplified by such strains as Actinomyces and Streptomyces and filamentous fungi. Suitable microorganisms are specifically noted in this text by their name and the American Type Culture Collection (ATCC) number assigned to them when deposited with a recognised International Depository Authority under the terms of the Budapest Treaty. Three new microorganisms were identified as being effective in this transformation and deposits were made with the American Type Culture Collection in Manassas, USA, under the terms of the Budapest Treaty. All three are gram positive filamentous bacteria, belonging to the Actinomycetales: Streptomyces species PTA-1685, Streptomyces cyaneus PTA-1686 and Streptomyces lydicus PTA-1687 and were cultivated on quarter strength ATCC172 agar slope.
The new microorganisms assigned Accession Numbers PTA-1685, PTA-1686 and PTA-1687 were all deposited with the ATCC at 10801 University Boulevard, Manassas, Va., 20110-2209, USA, on Apr. 11, 2000.
In a preferred embodiment X is selected from:
a) a 5- or 6-membered monocyclic aromatic ring optionally containing one or two heteroatoms, each independently selected from O, N and S; and
b) a C1-C6 alkylene group, straight chain or branched chain;
When X is as defined in (a) above, X may suitably be phenylene or a 5 or 6 membered aromatic heterocycle containing 1 heteroatom selected from O, N and S. Particularly suitably X may be phenylene or a 6 member aromatic heterocycle containing 1 heteroatom selected from O, N and S. Most suitably X is phenylene or pyridylene.
When X is as defined in (b) above, X may suitably be a C2-4 alkylene group, straight chain or branched chain. Most suitably X is ethylene or propylene.
It is believed that this process is specific to the oxidation of alkyl groups, attached directly or via a linker to a sulfonamide moiety, and that this reaction will be tolerated by a wide range of R groups. Accordingly R may encompass all organic radicals. Preferably, R is a 5- or 6-membered monocyclic aromatic ring optionally containing one or two heteroatoms, each independently selected from O, N and S, said ring being optionally further substituted. Particularly preferred is a 5- or 6-membered monocyclic aromatic ring containing two heteroatoms, each independently selected from O and N, said ring being optionally further substituted. Most preferred are substituted pyrazoles, substituted pyrimidines and substituted isoxazoles. The invention has been utilised with a variety of differing R substituents; suitable R substituents include: 
One embodiment of the present invention is a process for making compounds of formula (I) when Z is xe2x80x94CH2OH, Y is xe2x80x94C(CH3)2xe2x80x94 and X and R are as defined for formula
According to this embodiment, preferably X is phenylene, pyridylene, ethylene or propylene; most preferably X is 1,4-phenylene.
According to this embodiment R is preferably selected from one of:
3-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-1-methyl-4-p-tolylpyrazol-5-yl
4-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-5-p-tolylpyrimidin-6-yl
4-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(2-pyrimidinyl)pyrimidin-6-yl
4-methoxy-5-(2-methoxyphenoxy)-2-(2-pyrimidinyl)pyrimidin-6-yl
3-(2-ethoxyethoxy)-1-methyl-4-p-tolylpyrazol-5-yl
4-(1,3-benzodioxol-5-yl)-3-[2-(5-chloropyrimidin-2-yl)oxyethoxy]-1-methylpyrazol-5-yl
4-(1,3-benzodioxol-5-yl)-3-(2-hydroxyethoxy)-1-methylpyrazol-5-yl
4-(1,3-benzodioxol-5-yl)-3-[2-(5-bromopyrimidin-2-yl)oxyethoxy]isoxazol-5-yl
Most preferably R is 4-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-5-p-tolylpyrimidin-6-yl.
In this embodiment of the invention, micro-organisms particularly suitable for use in the process include: Streptomyces lavendulae ATCC14159, Streptomyces sp. PTA-1685, Streptomyces cyaneus PTA-1686, Streptomyces lydicus PTA-1687, Streptomyces griseus ATCC55070, Streptomyces griseolus ATCC11796, Amyclatopsis orientalis ATCC19795, Streptomyces griseus subsp. griseus ATCC13273, Streptomyces argentolus ATCC11009, Nocardia meditteranei ATCC21271, Streptomyces fumanus ATCC19904, Amycolata autotrophica ATCC35203, Streptomyces rimosus subsp. rimosus ATCC10970, Streptomyces griseus subsp. griseus ATCC10137, Streptomyces sp. ATCC31273, Cunninghamella echinulata var. elegans ATCC8688a, Mortierella isabellina ATCC42613, Verticillium lecanii ATCC60540, Mucor circinelloides ATCC7941, Cunninghamella echinulata var. echinulata ATCC36190, Syncephalastrum racemosum ATCC18192, Beauvaria sulphurescens ATCC7159, Absidia pseudocylindrospora ATCC24169, Amycolata autotrophica ATCC13181, Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC19067, Bacillus megaterium ATCC14581, Bacillus megaterium ATCC13368, Rhodococcus sp. ATCC19070, Actinomyces sp. ATCC53828, Bacillus subtilis ATCC55060, Pseudomonas putida ATCC17453, Pseudomonas putida ATCC49451, Bacillus sphaericus ATCC10208, Rhizopus oryzae ATCC11145, Absidia blakesleeeana ATCC10148a, Sepedonium chrysospermum ATCC13378, Alcaligenes eutrophus ATCC17697, Streptomyces galilaeus ATCC31133, Actinoplanes missouriensis ATCC23342, Actinoplanes missouriensis ATCC14538, Streptomyces peucetius subsp. caesius ATCC27952, Streptomyces lincolnensis ATCC25466, Streptomyces bambergiensis ATCC13879, Streptomyces argillaceus ATCC12956, Streptomyces albogriseolus ATCC31422, Streptomyces rutgersensis ATCC3350, Corynebacterium hydrocarboxydans ATCC21767, Streptomyces fradiae ATCC 10745, Streptomyces hydrogenans ATCC19631, Unidentified bacterium ATCC13930, Actinoplanes sp. ATCC53771, Thamnidium elegans ATCC18191, Aspergillus terreus ATCC10020, Curvularia lunata ATCC13432, Emericella unguis ATCC13431, Epicoccum humicola ATCC12722, Rhodococcus chlorophenolicus ATCC49826, Aspergillus ochraceus ATCC18500, Pithomyces cynodontis ATCC26150, Streptomyces roseochromogenes ATCC13400, Streptomyces griseus subsp. autotrophica ATCC53668, Streptomyces griseus subsp. griseus ATCC23337, Absidia repens ATCC14849 and Aspergillus alliaceus ATCC10060.
Preferred microorganisms for this embodiment where R is 3-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-1-methyl-4-p-tolylpyrazol-5-yl include: Syncephalastrum racemosum ATCC18192, Streptomyces sp. PTA-1685, Streptomyces lavendulae ATCC14159, Streptomyces cyaneus PTA-1686, Streptomyces griseus ATCC55070, Amycolatopsis orientalis ATCC19795, Streptomyces argentolus ATCC11009, Nocardia meditteranei ATCC21271, Streptomyces fumanus ATCC19904, Streptomyces rimosus subsp. rimosus ATCC10970, Streptomyces griseus ATCC10137, Cunninghamella echinulata ATCC8688a, Mortierella isabellina ATCC42613, Verticillium lecanii ATCC60540, Mucor circinelloides ATCC7941 and Cunninghamella echinulata ATCC 10028b.
Preferred microorganisms for this embodiment where R is 4-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-5-p-tolylpyrimidin-6-yl include: Streptomyces lavendulae ATCC14159, Streptomyces sp. PTA-1685, Streptomyces cyaneus PTA-1686, Streptomyces lydicus PTA-1687, Streptomyces griseus ATCC55070, Streptomyces griseolus ATCC11796, Amyclatopsis orientalis ATCC19795, Streptomyces griseus subsp. griseus ATCC13273, Streptomyces fumanus ATCC19904, Amycolata autotrophica ATCC35203, Streptomyces griseus subsp. griseus ATCC10137, Streptomyces sp. ATCC31273, Mortierella isabellina ATCC42613, Veticillium lecanii ATCC60540, Mucor circimelloides ATCC7941, Cunninghamella achinulata var. echinulata ATCC36190, Syncephalastrum racemosum ATCC18192, Amycolata autotrophica ATCC13181, Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC19067, Bacillus megaterium ATCC14581, Bacillus megaterium ATCC13368, Rhodococcus sp. ATCC19070, Actinomyces sp. ATCC53828, Bacillus subtilis ATCC55060, Pseudomonas putida ATCC49451, Bacillus sphaericus ATCC10208, Rhizopus oryzae ATCC11145, Absidia blakesleeeana ATCC10148a, Sepedonium chrysospermum ATCC13378, Alcaligenes eutrophus ATCC17697, Streptomyces galilaeus ATCC31133, Actinoplanes missouriensis ATCC23342, Actinoplanes missouriensis ATCC14538, Streptomyces peucetius subsp. caesius ATCC27952, Streptomyces lincolnensis ATCC25466, Streptomyces bambergiensis ATCC13879, Streptomyces argillaceus ATCC12956, Streptomyces albogriseolus ATCC31422, Streptomyces rutgersensis ATCC3350, Corynebacterium hydrocarboxydans ATCC21767, Streptomyces fradiae ATCC10745, Streptomyces hydrogenans ATCC19631, Unidentified bacterium ATCC13930, Actinoplanes sp. ATCC53771, Thamnidium elegans ATCC18191, Aspergillus terreus ATCC10020, Curvularia lunata ATCC13432, Emericella unguis ATCC13431 Epicoccum humicola ATCC12722, Rhodococcus chlorophenolicus ATCC49826, Aspergillus ochraceus ATCC18500, Streptomyces roseochromogenes ATCC13400, Streptomyces griseus subsp. autotrophica ATCC53668, Streptomyces griseus subsp. griseus ATCC23337, Absidia repens ATCC14849 and Aspergillus alliaceus ATCC10060.
Preferred microorganisms for this embodiment where R is 4-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(2-pyrimidinyl)pyrimidin-6-yl include: Streptomyces rimosus subsp. rimosus ATCC10970, Streptomyces fumanus ATCC19904, Streptomyces argentolus ATCC11009, Bacillus megaterium ATCC14538, Streptomyces griseus ATCC13273, Streptomyces griseus ATCC10137, Streptomyces griseolus ATCC11796, Streptomyces lavendulae ATCC14159, Streptomyces cyaneus PTA-1686, Streptomyces sp. PTA-1685, Amycolata autotrophica ATCC13181, Amycolata autotrophica ATCC35203 and Mortierella isabellina ATCC42613.
Preferred micro-organisms for this embodiment where R is 4-(1,3-benzodioxol-5-yl)-3-[2-(5-bromopyrimid-2-yl)oxyethoxy]isoxazol-5-yl include: Streptomyces rimosus subsp. rimosus ATCC10970, Streptomyces fumanus ATCC19904, Streptomyces argentolus ATCC11009, Bacillus megaterium ATCC14538, Streptomyces griseus ATCC13273, Streptomyces griseus ATCC10137, Streptomyces griseolus ATCC11796, Streptomyces lavendulae ATCC14159, Streptomyces cyaneus PTA-1686, Streptomyces sp. PTA-1685, Amycolata autotrophica ATCC13181, Amycolata autotrophica ATCC35203 and Mortierella isabellina ATCC42613.
Another embodiment of this aspect of the present invention is a process for making compounds of formula I when Z is xe2x80x94CH2OH, Y is xe2x80x94CH(CH3)xe2x80x94 and X and R are as defined in formula (I).
According to this embodiment, preferably X is phenylene, pyridylene, ethylene or propylene; most preferably X is 2,5-pyridylene, wherein Y is at the 2-position.
According to this embodiment, R is preferably 4-(1,3-benzodioxol-5-yl)-3-[2-(5-chloropyrimidin -2-yl)oxyethoxy]-1-methylpyrazol-5-yl.
Micro-organisms particularly suitable for use in this embodiment include: Streptomyces lavendulae ATCC14159, Streptomyces sp. PTA-1685, Streptomyces cyaneus PTA-1686, Streptomyces lydicus PTA-1687, Streptomyces griseus ATCC55070, Streptomyces griseolus ATCC11796, Amyclatopsis orientalis ATCC19795, Streptomyces griseus subsp. griseus ATCC13273, Streptomyces argentolus ATCC11009, Nocardia meditteranei ATCC21271, Streptomyces fumanus ATCC19904, Amycolata autotrophica ATCC35203, Streptomyces rimosus subsp. rimosus ATCC10970, Streptomyces griseus subsp. griseus ATCC10137, Streptomyces sp. ATCC31273, Cunninghamella echinulata var. elegans ATCC8688a, Mortierella isabellina ATCC42613, Verticillium lecanii ATCC60540, Mucor circinelloides ATCC7941, Cunninghamella echinulata var. echinulata ATCC36190, Syncephalastrum racemosum ATCC18192, Beauvaria sulphurescens ATCC7159, Absidia pseudocylindrospora ATCC24169, Amycolata autotrophica ATCC13181, Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC19067, Bacillus megaterium ATCC14581, Bacillus megaterium ATCC13368, Rhodococcus sp. ATCC19070, Actinomyces sp. ATCC53828, Bacillus subtilis ATCC55060, Pseudomonas putida ATCC17453, Pseudomonas putida ATCC49451, Bacillus sphaericus ATCC10208, Rhizopus oryzae ATCC11145, Absidia blakesleeeana ATCC10148a, Sepedonium chrysospermum ATCC13378, Alcaligenes eutrophus ATCC17697, Streptomyces galilaeus ATCC31133, Actinoplanes missouriensis ATCC23342, Actinoplanes missouriensis ATCC14538, Streptomyces peucetius subsp. caesius ATCC27952, Streptomyces lincolnensis ATCC25466, Streptomyces bambergiensis ATCC13879, Streptomyces argillaceus ATCC12956, Streptomyces albogriseolus ATCC31422, Streptomyces rutgersensis ATCC3350, Corynebacterium hydrocarboxydans ATCC21767, Streptomyces fradiae ATCC10745, Streptomyces hydrogenans ATCC19631, Unidentified bacterium ATCC13930, Actinoplanes sp. ATCC53771, Thamnidium elegans ATCC18191, Aspergillus terreus ATCC10020, Curvularia lunata ATCC13432, Emericella unguis ATCC13431, Epicoccum humicola ATCC12722, Rhodococcus chlorophenolicus ATCC49826, Aspergillus ochraceus ATCC18500, Pithomyces cynodontis ATCC26150, Streptomyces roseochromogenes ATCC13400, Streptomyces griseus subsp. autotrophica ATCC53668, Streptomyces griseus subsp. griseus ATCC23337, Absidia repens ATCC14849 and Aspergillus alliaceus ATCC10060.
The oxidation of the alkyl group to the corresponding hydroxy derivative using a P450 enzyme may be allowed to continue onwards to the carboxylic acid. No modification to the process is required other than that the process be given sufficient time to further oxidise the substrate. It will be appreciated that by careful manipulation of the reaction conditions, one may selectively isolate a product at the desired oxidation level and maximise the yield of the desired product. Further, the nature of the R substituent may influence the speed of the reaction and hence the yields of the desired products. Whilst all P450 enzymes are considered suitable for conducting the disclosed invention, certain microorganisms will be particularly suitable for specific levels of oxidations.
Another embodiment of the present invention is a process for making compounds of formula (I) when Z is xe2x80x94COOH, Y is xe2x80x94C(CH3)2xe2x80x94 and X and R are as defined in formula (I).
In this embodiment, preferably X is phenylene, pyridylene, ethylene or propylene; most preferably X is 1,4-phenylene.
According to this embodiment, R is preferably selected from one of;
3-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-1-methyl-4-p-tolylpyrazol-5-yl
4-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-5-p-tolylpyrimidin-6-yl
1-methyl-3-(2-methoxyethoxy)-4-p-tolylpyrazol-5-yl
3-(2-ethoxyethoxy)-1-methyl-4-p-tolylpyrazol-5-yl.
Most preferably R is 4-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-5-p-tolylpyrimidin-6-yl.
Microorganisms particularly suitable for use in this embodiment include: Amycolata autotrophica ATCC 35203, Nocardia meditteranei ATCC21271, Amycolatopsis orientalis ATCC19795 Streptomyces griseolus ATCC11796, Streptomyces rimosus subsp. rimosus ATCC10970 and Nocardia meditteranei ATCC21271.
Preferred microorganisms for this embodiment where R is 3-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-1-methyl-4-p-tolylpyrazol-5-yl include: Amycolata autotrophica ATCC 35203, Nocardia meditteranei ATCC21271 and Amycolatopsis orientalis ATCC19795
Preferred micro-organisms for this embodiment where R is 4-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-5-p-tolylpyrimidin-6-yl include: Streptomyces griseolus ATCC 11796, Streptomyces rimosus subsp. rimosus ATCC10970 and Nocardia meditteranei ATCC21271.
A further embodiment of the present invention is a process for making compounds of formula (I) when Z is xe2x80x94COOH, Y is xe2x80x94CH(CH3)xe2x80x94 and X and R are as defined in formula (I).
According to this embodiment, preferably X is phenylene, pyridylene, ethylene or propylene; most preferably X is 2,5-pyridylene.
Microorganisms particularly suitable for use in this embodiment include Amycolata autotrophica ATCC 35203, Nocardia meditteranei ATCC21271, Amycolatopsis orientalis ATCC19795 Streptomyces griseolus ATCC11796, Streptomyces rimosus subsp. rimosus ATCC10970 and Nocardia meditteranei ATCC21271.
The introduction of a functional group into a complex molecule that already possesses a wide range of functionality is extremely difficult. Unless one has absolute specificity it is likely that the reaction will be accompanied by a host of competing side reactions. The effect of this is to produce the desired compound in a smaller yield and to provide difficulties in extracting and purifying the desired molecule. These problems are particularly acute in the case of oxidising a Cxe2x80x94H bond. This is almost impossible to achieve selectively using standard chemical means.
To avoid this problem, the skilled synthetic chemist will employ a careful retrosynthetic analysis of the synthetic target. The synthetic strategy will involve avoiding side reactions and will usually involve a protecting group strategy. In avoiding conflicting side reactions, the chemist will be forced to add extra reaction steps to his synthesis, e.g. protection and deprotection.
These extra steps are undesirable. There are the additional costs of the reagents, solvents and other manufacturing costs. The extra steps will also depress the overall yield, again adding to the cost of the final product. Further there are the problems associated with purifying the compound after each step and disposing of any pollutants.
The process of the present invention solves these problems by introducing a hydroxy or carboxy moiety into a molecule with great specificity in one step with high yield. So specific is the process, that such moieties may be introduced at a very late stage, into a molecule with a multiplicity of other functionality. The advantage of this process are that fewer synthetic steps are required leading to lower costs in reagents, solvents and production costs. Further there will be fewer pollutants resulting in less environmental damage and clean up costs. The process is also suitable for production on an industrial scale.
Microorganisms containing these P450 cytochrome enzymes suitable for use in this invention may be obtained from a variety of sources. They may be isolated from various soil samples, then grown on various agars as described in the ATCC Catalogue of Bacteria and Bacteriophages, 18th Edition, 1992, or the ATCC Catalogue of Fungi and Yeasts, 17th Edition, 1987.
The processes of the invention may be carried out in any suitable aqueous fermentation medium.
It will be appreciated that the process of the present invention may be conducted in the presence of various additives such as solubilisers such as alcohols and dipolar aprotic solvents, e.g. methanol and dimethylsulfoxide.
The wide range of functionality able to tolerate the process of the present invention is exemplified in the following examples;
High performance liquid chromatography (HPLC) retention times and UV spectra were recorded using a Hewlett-Packard 1090 LUSI diode-array spectrophotometer (method A). All NMR spectra were measured in CDCl3 or MeOD by an Inova 300 MHz or 400 MHz spectrometer unless otherwise indicated and peak positions are expressed in parts per million (ppm) downfield from tetramethylsilane. The peak shapes are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br broad. High resolution MS data was acquired on a AutoSpecQ with electrospray ionisation (ESI) or thermospray ionisation (TSPI) using a PEG reference (or on a Bruker Apex II FTMS with ESI where indicated).
Infra red data was collected on a Perkin Elmer Paragon 1000 FT-IR using conventional techniques such as a polyethylene (PE) film or as a NUJOL dispersion on sodium chloride (NaCl) discs. Peak positions are expressed in cmxe2x80x941.
HPLC-MS data was acquired using a Hewlett-Packard 1090M liquid chromatograph interfaced to a VG platform II mass spectrometer equipped with an ES source (method B).
Use is made of the following fermentation media.